Ultrasound is a valuable diagnostic imaging technique for studying various areas of the body including, for example, the vasculature, such as tissue microvasculature. Ultrasound provides certain advantages relative to other diagnostic techniques. For example, diagnostic techniques involving nuclear medicine and X-rays generally results in exposure of the patient to ionizing electron radiation. Such radiation can cause damage to subcellular material, including deoxyribonucleic acid (DNA), ribonucleic acid (RNA) and proteins. Ultrasound does not involve such potentially damaging radiation. In addition, ultrasound is relatively inexpensive as compared, for example, to computed tomography (CT) and magnetic resonance imaging (MRI), which require elaborate and expensive equipment.
Ultrasound involves the exposure of a patient to sound waves. Generally, the sound waves dissipate due to absorption by body tissue, penetrate through the tissue or reflect off of the tissue. The reflection of sound waves off of tissue, generally referred to as backscatter or reflectivity, forms the basis for developing an ultrasound image. In this connection, sound waves reflect differentially from different body tissues. This differential reflection is due to various factors, including, for example, the constituents and the density of the particular tissue being observed. The differentially reflected waves are detected, typically with a transducer that can detect sound waves having a frequency of one megahertz (MHz) to ten MHz. The detected waves can be integrated, quantitated and converted into an image of the tissue being studied.
Ultrasound imaging techniques typically involve the use of contrast agents. Contrast agents are used to improve the quality and usefulness of images which are obtained via ultrasound. Exemplary contrast agents include, for example, suspensions of solid particles, emulsified liquid droplets, and gas-filled bubbles. See, e.g., Hilmann et al., U.S. Pat. No. 4,466,442, and published International Patent Applications WO 92/17212 and WO 92/21382.
The quality of images produced from ultrasound has improved significantly. Nevertheless, further improvement is needed, particularly with respect to images involving vasculature in tissues that are perfused with a vascular blood supply. Accordingly, there is a need for improved ultrasound techniques, including improved contrast agents, which are capable of providing medically useful images of the vasculature and vascular-related organs.
The reflection of sound from a liquid-gas interface is extremely efficient. Accordingly, bubbles, including gas-filled bubbles, are useful as contrast agents. The term "bubbles", as used herein, refers to vesicles which are generally characterized by the presence of one or more membranes or walls surrounding an internal void that is filled with a gas or a precursor thereto. Exemplary bubbles include, for example, liposomes, micelles and the like. As discussed more fully hereinafter, the effectiveness of bubbles as contrast agents depends upon various factors, including, for example, the size, elasticity and/or stability of the bubble.
With respect to the effect of bubble size, the signal that is reflected off of a bubble is a function of the radius (r.sup.6) of the bubble (Rayleigh Scatterer). Thus, a bubble having a diameter of 4 micrometer (.mu.m) possesses about 64 times the scattering ability of a bubble having a diameter of 2 .mu.m. Thus, generally speaking, the larger the bubble, the greater the reflected signal.
However, bubble size is limited by the diameter of capillaries through which the bubbles must pass. Contrast agents which comprise bubbles having a diameter of greater than 10 .mu.m are generally dangerous since microvessels may be occluded. Accordingly, it is desired that greater than about 99% of the bubbles in a contrast agent have a diameter of less than 10 .mu.m. Mean bubble diameter is important also, and should be greater than 1 .mu.m, with greater than 2 .mu.m being preferred. The volume weighted mean diameter of the bubbles should be about 7 to 10 .mu.m.
Bubble elasticity is also important because highly elastic bubbles can deform, as necessary, to "squeeze" through capillaries. This decreases the likelihood of occlusion. In addition, resonance is more easily induced in bubbles having enhanced elasticity. This can be advantageous in that resonating bubbles typically generate sound emissions at frequencies in the subharmonic regime (based on multiples of 0.5) or in the supra- or ultraharmonic regime (based on multiples of 2). The supraharmonic regime, including second harmonic imaging, is desirable in ultrasound since background noise is substantially eliminated. Elastic bubbles can therefore be used to produce desirable second harmonic images.
The effectiveness of a contrast agent involving bubbles is also dependent on the bubble concentration. Generally, the higher the bubble concentration, the greater the reflectivity of the contrast agent.
Another important characteristic which is related to the effectiveness of bubbles as contrast agents is bubble stability. As used herein, particularly with reference to gas-filled bubbles, "bubble stability" refers to the ability of bubbles to retain gas entrapped therein after exposure to a pressure greater than atmospheric pressure. To be effective as contrast agents, bubbles generally need to retain an amount of the entrapped gas in vivo. It is also highly desirable that, after release of the pressure, the bubbles return to their original size. This is referred to generally as "bubble resilience."
Bubbles which lack desirable stability provide poor contrast agents. If, for example, bubbles release the gas entrapped therein in vivo, reflectivity is diminished. Similarly, the size of bubbles which possess poor resilience will be decreased in vivo, also resulting in diminished reflectivity.
The stability of bubbles disclosed in the prior art is generally inadequate for use as contrast agents. For example, the prior art discloses bubbles, including gas-filled liposomes, which comprise lipoidal walls or membranes. See, e.g., Ryan et al., U.S. Pat. Nos. 4,900,540 and 4,544,545; Tickner et al., U.S. Pat. No. 4,276,885; Klaveness et al., WO 93/13809 and Schneider et al., EPO 0 554 213 and WO 91/15244. The stability of the bubbles disclosed in these references is poor in that as the solutions in which the bubbles are suspended become diluted, for example, in vivo, the walls or membranes of the bubbles are thinned. This results in a greater likelihood of rupture of the bubbles.
Various attempts have been made to improve bubble stability. Such attempts have included, for example, the preparation of bubbles in which the membranes or walls thereof are apparently strengthened via crosslinking. See, e.g., Giddey et al., U.S. Pat. No. 5,310,540 and Klaveness et al., WO 92/17212, in which there are disclosed bubbles which comprise proteins crosslinked with crosslinking agents.
Prior art techniques for stabilizing bubbles, including the use of crosslinked materials, suffer from various drawbacks. For example, the crosslinked materials described, for example, in Giddey et al., U.S. Pat. No. 5,310,540 and Klaveness et al., WO 92/17212, lack biocompatibility or possess unknown metabolic fates. Added costs are also incurred with the use of additional materials and process steps necessary for crosslinking. In addition, crosslinking can impart rigidity to the membranes or walls of the bubbles. This results in bubbles having reduced elasticity and, therefore, a decreased ability to deform and pass through capillaries. Thus, there is a greater likelihood of occlusion of vessels with prior art contrast agents that are stabilized via crosslinking.
Accordingly, new and/or better stabilized contrast agents and methods for providing same are needed. The present invention is directed to this, as well as other, important ends.